FAU (1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-uracil) has been studied as a potential therapeutic and PET imaging agent. The mechanism by which FAU exerts cytotoxicity is not yet well understood but is likely due to either inhibition of DNA or RNA synthesis and/or toxicity once incorporated into DNA. FAU was designed to take advantage of high thymidylate synthase (TS) expression levels observed in breast, colorectal, head and neck cancers that become resistant to 5-fluorouracil (5FU) treatment. Once FAU is taken up into cells, it is phosphorylated to FAU monophosphate (FAUMP) by thymidine kinase (TK) and then methylated by thymidylate synthase to FMAUP (Collins et al., 1999). After further phosphorylation, FMAU-TP is utilized in DNA synthesis (Klecker et al., 1994). So instead of inhibiting TS, an anti-neoplastic drug targeting strategy, FAU is activated by the high TS levels into a cytotoxic drug. See, also U.S. Pat. Nos. 6,682,715; 6,667,314; and 6,703,374.
Studies have demonstrated that [C-14]FAU is incorporated into DNA as 2′-F-ara-5-methyl-deoxyuridine (FMAU), and this appears to be its mode of cytotoxicity (Collins et al., 1999). After incubation of cell lines with FAU or FMAU, the percentage of thymidine replaced correlated with growth inhibition (Collins et al., 1999). These in vitro studies were further supported by in vivo studies in mice showing the incorporation of FAU into DNA as FMAU (Wang et al., 2002). This males FAU a particularly attractive drug, since tumors that are resistant to 5-fluorouracil (5FU) and other TS inhibitors are expected to be more sensitive to FAU toxicity. However, preliminary studies of FAU as an imaging agent demonstrated that it was poorly concentrated in normal proliferating tissues such as marrow in dogs or humans, although modest increased uptake was seen in some tumors (Sun, 2003), potentially limiting its therapeutic effectiveness. In concurrence with this observation, in vitro TK assays showed that FAU is a very poor substrate for mammalian cytologic thymidine kinase (TK1) relative to thymidine or FMAU (Sun et al., 2003). This preliminary data suggests that FAU would be of limited efficacy as an imaging or therapeutic agent since its activation is inhibited at the first phosphorylation step. While there is no evidence that TS activation would not become another hurdle, a recent report (Eiseman et al., 2004) showed that TS activation is the most determinant step for incorporation of FAU into DNA. In principle, administration of 5′-phosphate would aid in overcoming this problem. However, because phosphates are strongly acidic and thus negatively charged at physiological pH, they are too hydrophilic to penetrate the lipid-rich cell membranes. Furthermore, extracellular phosphatases are likely to remove the phosphate. Therefore, there is a need for strategies to circumvent the poor phosphorylation of FAU by mammalian thymidine kinases.